P53融合蛋白的原核表达及其单克隆抗体的制备

目的 为研制出能特异与Ｐ＾５３结合的单克隆抗体，使对Ｐ＾５３的检测得到更为广泛的应用。方法 用ＰＣＲ技术扩增编码人Ｐ＾５３Ｎ－端１８０个氨基酸的ＤＮＡ片段并将其克隆在谷胱苷肽转移酶（ＧＳＴ）表达质粒Ｐ＾ＧＥＸ－２Ｔ中。用该重组质粒转化大肠杆菌ＪＭ１０９，并经异丙基β－Ｄ硫代半乳糖苷（ＩＰＴＧ）诱导产生Ｐ＾５３－ＧＳＴ融合蛋白。用Ｐ＾５３－ＧＳＴ免疫ＢＡＬＢ／ｃ小鼠。常规细胞融合，间接酶联免疫吸附试

EXPRESSION OF P53 FUSION PROTEIN IN PROKARYOTE AND PREPARATION OF MONOCLONAL ANTIBODY TO P53

Objective We tried to probe the antitumor mechanisms of new antitumor drug norcantharidin(NCTD) on human hepatoma (BEL 7402) cells. Methods By morphological observation and DNA agarose gel electrophoresis, we examined the characteristics of NCTD on human hepatoma cells, then determined the expression of oncoprotein Bcl 2 by immunocytochemical and Western blotting analysis in this process. Results Treated with 10mg/L NCTD for 24h, some human hepatoma cells condensed with membrane budding, the nuclear chromatin compaction and membrane enclosed apoptotic bodies formation, and fragments that were multiples of units comprising about 200 base pairs were detected by agarose gel electrophoresis, a characteristic ladder being evident when ethidium bromide stained gels were viewed in ultraviolet light. The expression of oncoprotein Bcl 2 was proved decreasing in human hepatoma cells at this time. Conclusion The new antitumor drug NCTD can induce apoptosis in human hepatoma cells, and the declining expression of oncoprotein Bcl 2 might involve in this process.